The 2014 World Health Organization (WHO) Classification of cervical NETs has been updated to adopt the terms of low-grade NETs (including grade 1 and grade 2, encompassing what were previously called carcinoids and atypical carcinoids) and high-grade neuroendocrine carcinoma (encompassing what were previously called small cell carcinoma and large cell carcinoma) [
2]. Small cell carcinoma of the cervix represents the most common cervical NET, followed by cervical large cell neuroendocrine carcinoma and low-grade NETs [
16,
17]. Grade 2 or atypical carcinoid tumors represent a proportion of the rarest entity of cervical NETs. A PubMed search revealed that only 17 grade 2 NETs had been reported [
3‐
6]. In this study, we present a series of three cervical grade 2 NETs with their clinicopathological and molecular features.
The morphology of our cases did not fall beyond the description of existing studies. Grade 2 neuroendocrine tumors are well-differentiated. The histo-morphological features of these tumors are similar to the features of lung and gastrointestinal tract neuroendocrine neoplasms. Grade 2 neuroendocrine tumors take the form of nested organoid, columnar, insular or trabecular patterns. The tumor cells are epithelioid or spindled and feature relatively abundant eosinophilic cytoplasm and nuclei with dense granular chromatin, and at times, pleomorphic and inconspicuous nucleoli [
2]. Necrosis can be discovered in focal areas, and the mitotic count is 2–10 mitotic figures per 10 high-power fields. The tumors of our 3 cases also showed nest-like, trabecular, sheet-like, “single file” strands or rosette-like structures, and lympho-vascular invasion was also observed. A total of 2 to 5 mitotic figures in every 10 high-power fields was observed within the tumors of our 3 cases, and necrotic foci were only observed in case 1 who died because of metastases to the liver and bone.
Grade 2 NETs of the uterine cervix are rare and probably underdiagnosed or misdiagnosed. Immunohistochemistry is useful for detecting neuroendocrine differentiation. Immunohistochemically, grade 2 NETs are diffuse and positive for synaptophysin (Syn), chromogranin A (Cg A) and CD56, and the Ki-67 proliferation index indicates the malignant characteristics of neuroendocrine tumors. In our cases, neuroendocrine markers including Syn, Cg A and CD56 were positive, and the Ki-67 proliferation index was between 2% and 7%, which was similar to the grade 2 NETs (atypical carcinoids) of the cervix reported previously [
3‐
6]. We also stained for P40, P63, and CK7 markers for the differential diagnosis of poorly differentiated squamous cell carcinoma and adenocarcinoma, as the histology could be confusing in the case of grade 2 NETs. The patients in our study had a thorough check-up using MRT, CT, and ultrasound. No tumors were found in other parts of the body. Therefore, the detailed and rigorous clinicopathological analysis made metastatic grade 2 NETs unlikely.
The molecular and genetic treatment of well-differentiated NETs occurring in the pancreas, gastrointestinal tract and lung has been dramatically improved by agents targeting the multiple receptor tyrosine kinase (RTK), vascular endothelial growth factor (VEGF) or the mammalian target of rapamycin (MTOR). The RTK inhibitor sunitinib was approved for the treatment of well-differentiated pancreatic NETs in 2011, and in 2016, the MTOR inhibitor everolimus was approved for well-differentiated NETs occurring in the gastrointestinal tract and the lung [
7,
8]. Other biologics such as the VEGF-A inhibitor bevacizumab have also provided promising clinical solutions [
7]. To confirm whether there are similar genetic changes occurring in grade 2 NETs compared with those of the pancreas, gastrointestinal tract and lung, the molecular and genetic changes in cervical grade 2 tumors of our three cases were studied. Unfortunately, the alterations of MTOR and the RTK pathway were not observed in the 3 cases, whereas other genetic changes were observed, such as the non-frameshift insertion of MAGI1 (cases 1 and 2) and SLC45A3 (cases 1 and 3). The case 1 with the non-frameshift insertion of both MAGI1 and SLC45A3 was died 27 months after the operation because of metastases to the liver and bone, while the molecular mechanism of MAGI1 and SLC45A3 to the cervical grade 2 tumor of this case is sill unknown and need to further study. The missense mutations, the non-frameshift deletion and the frameshift insertion in one of the three cases were also detected (as shown in Fig. 7). However, the changes and significance of these genetic changes for the treatment of cervical grade 2 neuroendocrine tumors have not been reported previously. Membrane-associated guanylate kinase inverted 1 (MAGI1), as a member of the membrane-associated guanylate kinase family, was downregulated in diverse cancers and was a tumor suppressor in colorectal cancer, hepatocellular carcinoma, cervical cancer and gastric cancer in previous reports [
18‐
22]. Christian K et al. reported that MAGI-1 is a sensitive proteolytic substrate for both the HPV-16 and HPV-18 E6 oncoproteins, and its expression is always lost in HPV-positive cervical cancer cells [
22]. Their findings also suggested that E6-mediated inhibition of MAGI-1 function contributes to HPV pathology by perturbing tight junction assembly with concomitant stimulation of proliferation and inhibition of apoptosis, and the restoration of MAGI-1 expression in HPV-positive cervical tumor cells could induce cell growth arrest and apoptosis [
22]. Our study showed the non-frameshift insertion of MAGI1 in two cases and the immunohistochemically positive P16 indicated HPV infection of all our 3 cases [
23], while the relation of MAGI1 and the HPV E6 oncoproteins was unknown and needs to further study in our cases. A study reported that the rearrangement of ERG with SLC45A3 and the loss of SLC45A3 expression were one of the aggressive pathways of prostate cancer progression [
24]. The non-frameshift insertion of SLC45A3 was shown in two of our cases, and the study of the relationship between the non-frameshift insertion of SLC45A3 and the cervical grade 2 NETs is recommended. The mutation spectrum of the three cases presented did not overlap with an earlier study on the molecular profile of cervical NETs. Deyin X et al. reported next-generation sequencing based on a 637-gene panel of small cell neuroendocrine carcinoma of the uterine cervix in 10 cases and found the mutations of the gene changes of TP53, PIK3CA, KRAS, Erbb2, c-Myc, NOTCH1, BCL6, NCOA3, PTEN, RB1, BRCA1, BRCA2, and ARID1B; genetic alterations involving the MAPK, PI3K/AKT/mTOR, and TP53/BRCA pathways were also observed in their study [
25]. While We detected the mutations of FCGR3A, STK36, TNK2, DAXX, EZR, HIF1A, SEPT9, PMS1, UGT1A3, XPC, ITK, NOTCH4, IGF2R, AKAP9, EPHB4, RET, KAT6B, NUMA1, ADAMTS20, YES1, BCR, PDE4DIP, PAX8, MLH1, SDHA, MET, TET1, BRCA2, ASXL1, MAGI1, SLC45A3, EP400 based on a 560-gene panel of grade 2 NETs of the uterine cervix in 3 cases, which indicated that the cervical grade 2 NETs may be have their own unique gene alterations and regulations compared with those of cervical small cell neuroendocrine carcinoma. In-depth investigation in a larger cohort of genetic alterations could provide more useful information to guide the molecular characterization and potential individualized treatment of grade 2 cervical NETs.